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NMEICT-MHRD (Govt.
of India) Project on - Creation of e-Contents on Fermentation Technology
Module – 15: Batch and continuous sterilization process
Medium sterilization
Media sterilized by
1) Filtration,
2) Radiation,
3) Ultrasonic treatment,
4) Chemical treatment
5) Heat
Out of these methods, heat or steam is the most useful method for the sterilization of
fermentation media.
A number of factors influence the success of heat sterilization
1 The number and types of microorganisms present

2 The composition of the culture medium
3 The pH value and the size of the suspended particle
Filtration used for the sterilization of medium, which is exception for the medium containing
heat labile components.
Sterilization process i.e. killing of microbes by steam under pressure is a first-order chemical
reaction and, thus, may be written as.
-dN/dt = kN 1
Where
N is the number of viable organisms present,
t is sterilization treatment time,
K is the reaction rate constant of the reaction,or the specific death rate
Regardless of the volume of the batch, the minimum number of organisms to contaminate a
batch is one.
On integration of equation (5.1), the following expression obtained….
Project control No: RE-02091011297, Christ College, Rajkot, Gujarat, India

NMEICT-MHRD (Govt. of India) Project on - Creation of e-Contents on Fermentation Technology
Nt/No = e-kt (2)
No is the number of viable organisms present initially
Nt is the number of viable organisms present after a period treatment, t
On taking natural logarithms, equation (5.2) reduced to
ln(Nt/ No) = -kt (3)
kt= ln (N0/ Nt) (4)
The graphical representations of equations (2) and (3) illustrated in Fig. 5.1,
A plot of the natural logarithm of Nt/ Noagainst time yields a straight line, the slope of which
equals -k.
This kinetic description makes two predictions, which appear abnormal.
1. Sterile condition is achieved in an infinite time (i.e. Nt = 0)
2. After a definite time number of cells present will be less than one
The relationship displayed in Fig. 5.1 observed only for pure culture in certain metabolic state,
under ideal sterilization conditions.
The value of k also indicates physiological state of organisms with kind of species, but
dependent on the physiological form of the cell; for example, the endospores of the genus
Bacillus are far more heat resistant than the vegetative cells.

Richards (1968) produced a various graphs illustrating the deviation from theory, which
experienced in practice.
Figure 5.2shows the relation between bacterial endospore survivals versus time of treatment
The deviation in the above graph is due to the induction of spore germination by the heat and
moisture of the initial period of the sterilization process.
Fig. 5.2(a)early stages provide temperature for the growth of the spores hence the number of
viable cells increases in the initial stage and then there is killing of spores at the later stage.
In Fig. 5.2(b),activation balanced by spore death and in Fig. 5.2(c)activation is less than spore
death.
Figures 5.3 illustrate typical results of the sterilization of mixed cultures containing two species
with different heat sensitivity.

In Fig. 5.3a the population consists mainly of the less-resistant type where the initial decline
is due principally to the destruction of the less-resistant cell population and the later, less
rapid decline, is due principally to the destruction of the more resistant cell population
Figure 5.3b represents the reverse situation where the more resistant type predominates
and its presence disguises the decrease in the number of the less resistant type
Time for the sterilization is dependent on the type of the population. If the sensitive organisms
are more in number than whole culture sterilization will be equal to that of the sensitive culture.
However, if the number of the resistant organisms is more than the sterilization of whole culture
is equal to that of the sterilization of the resistant organisms.Now by considering that
contaminant may be by not a single type of organism but by different types of organisms.
Sterilization of media required destruction of all types of organisms. The destruction of
organisms in sterilization process given by the factor called Del factor.As first order reaction, as
temperature increases, reaction rate increases due to an increase in the reaction rate constant,
which, in case of the destruction of microorganisms is the specific death rate (k)?Thus, k is true
constant only under constant temperature conditions.
The relationship between temperature and reaction rate constant was demonstrated by Arhenius
and may be represented by the equation
D ln k = E
dT RT2
E – Activation energy
R – Gas constant
T – Absolute temperature
On Integration
K = A. e –E/RT
Therefore kt = A. t. e –E/RT
On taking natural logarithm
ln k = ln A – E/RT
Plot of ln k versus 1/T gives straight line
Such a plot is Arhenius plot and enable calculation of activation energy and prediction of the
reaction rate for any temperature.
Now lnNt/No = -kt
So, ln No/Nt = kt
Therefore ln No/Nt = A. t. e –E/RT
Deindoerfer and Humphrey (1959) used the term In No / Ntas a design criterion for sterilization,
which has been variously called the Del factor, Nabla factor and sterilization criterion
represented by the term ▼
Thus, the Del factor gives idea about fractional reduction in viable organism count produced by a
definite heat in particular time.
Now ▼= ln No/Nt
But from above equation ln No/Nt = A. t. e –E/RT
Thus ▼= A. t. e –E/RT
On rearranging equation
ln t = E/RT + ln (▼/A)
This graph used to obtain definite ▼ value with certain absolute temperature.
If we plot the graph from above line equation then we will get idea about time and absolute
temperature required to achieve sterilization.
According to Deindoerfer and Humphrey, Richards Banks and Corbett a risk factor of one batch
in a thousand contaminated frequently used in fermentation industries – that is, the final
microbial count in the medium after sterilization should be 10-3 viable cells.
To apply kinetics it is necessary to know the thermal death characteristics of all the taxa
contaminating the fermenter and unsterile medium, this is an impossibile and, therefore, the
assumption may be made that the only microbial contaminants present are spores of Bacillus
stearotheromphilus- that is, one of the most heat-resistant microbial types known.
Thus, by adopting B. stearothennophilus as the design organism a considerable safety factor built
into the calculations.
It should be remembered that B. stearotheromphilusis not always adopted as the design

organism.
If the most heat-resistant organism contaminating the medium ingredients is known, then it may
be advantageous to base the sterilization process on this organism.
Figure 5.5 illustrates the effect of increasing medium sterilization on the yield of product of
subsequent fermentations.

The initial rise in yield is due to some components of the medium made more available to the
process microorganism by the 'cooking effect' of a brief sterilization period (Richards, 1966).
During sterilization,the nutrient value of the medium decreasesdue to following Reactions.
Interactions between nutrient components of the medium
 Maillard-type browning reaction discoloration of the medium as well as deterioration

ofnutrient valuecaused by the reaction of carbonyl groups and amino groups from reducing
sugars, and amino acids and proteins respectively.
Degradation of heat labile components
 Certain vitamins, amino acids and proteins may be degraded during a steam sterilization
regime
 Thus heat labile compounds can be sterilized by filtration
 However, for the vast majority of fermentations these problems may be resolved by the
judicious choice of steam sterilization regime
The activation energy for thermal destruction of Bacillus steareothermophilusspores is more than
for thermal destruction of nutrient.

Thus, it would be advantageous to employ high temperature for shorter period to achieve desired
probability of sterility, yet causing minimum degradation of nutrients.
Batch sterilization is not possible, as high temperature cannot be kept for short period by this
method thus only solution to this problem is continuous stream sterilization
Advantages of continuous sterilization over batch sterilization
1. Betterprotectionof medium value

2. Ease of scale-up - discussed later
3. Easier automatic control
4. The decrease of flowability for steam
5. The reduction of sterilization cycle time
6. Under certain conditions, the decreasein corrosion of fermentor
Advantages of batch sterilization over continuous sterilization
1. Lesser assetsapparatusexpenditure
2. Less chance of contamination - processes require the aseptic inoculums transfer of the
sterile broth to the sterile vessel
3. Easier manual control
4. Easier to use with media having a high amount of solid material
The Design of Batch Sterilization Processes
Main aim of batch sterilization process is still to attain the necessary chance of getting sterility
with the least change in nutrient value of the medium. Continuous sterilization process is better
than batch sterilization process in avoiding the damage of nutrients than a continuous
sterilization process.The maximum temperature, which is possible in batch sterilization, is 121°C
therefore a method adopted so that medium exposed to this high temperature for a short
period.High temperature and short time sterilization attained by taking into consideration the
heating and cooling time of the batch sterilization.Deindoerfer and Humphrey (1959) offered a
method to evaluate the role of heating and cooling periods in sterilization process.
The following point should take into consideration for a batch sterilization process
1. How much temperature of the fermentation medium is increased during heating or

decreased during cooling periods of the batch sterilization
2. The initially number of micro-organisms in the medium
3. The thermal death rate of the selected organism

Requisite Del factor is design by knowing the initial number of organisms in the medium and the
danger of contamination.
Commonly accepted threat of contamination is one in 1000, which means number of living
organisms after time t is 0.001.
For example if any unsterile broth contain 1011 number of cells then Del factor for that situation
is 32.2
 However, the killing of cells take place during both the heating period and cooling period
of the sterilization process in addition to during holding period at 121°C
 So, Del factor can be
▼overall = ▼heating +▼holding +▼cooling
 Knowing the temperature and time required to reach that temperature during heating period
and cooling period of sterilization process it is possible to determine the overall Del factor
by these periods
 Thus, from the Del factors contributed by heating and cooling periods, it is possible to
estimate the holding time that may be required for overall Del factor
Batch sterilization Methods
The batch sterilization of the medium for fermentation achieved either in the fermentation vessel
or in a separate mash cooker. Richards (1966) considered the relative merits of in situ medium
sterilization and the use of a special vessel
Advantages of a separate medium sterilization vessel
 The medium sterilized in a cooker in a more concentrated form than would be used in the
fermentation and then diluted in the fermenter with sterile water prior to inoculation. This
would allow the construction of smaller cookers
 One cooker may be used to serve several fermenters and the medium may be sterilized as
the fermenters are being cleaned and prepared for the next fermentation, thus saving time
between fermentations
 In some fermentation, the medium is at its most viscous during sterilization and the power
requirements for agitation increased lessen by aeration. Fermenter equipped with a
powerful motor would provide sterile medium for several fermenters
 The fermenter spared the corrosion, which may occur with medium at high temperature.

Disadvantages of a separate medium sterilization vessel
 The cost of constructing a batch medium sterilizer is much the same as that for the
fermenter
 If a cooker serves a large number of fermenters complex pipe work would be necessary to
transport the sterile medium, with the inherent dangers of contamination
 Mechanical failure in a cooker supplying medium to several fermenters would render all
the fermenters temporarily redundant (unneeded). The provision of contingency equipment
may be prohibitively costly
The design of continuous sterilization process
 The plan of continuous sterilization system may be advanceprecisely the same as batch
sterilization systems
 In continuous sterilization medium is heated to reach to the sterilization temperature
(121oC), holding this temperature to particular period of time and then cooling the medium
to reach to the temperature of the fermentation process
 The temperature of the medium is increased in a continuous heat exchanger and is
maintained for the holding time in an shieldingwinding holding coil
 The extent of the holding time is stated by the coil length and the medium streamspeed
 The medium after holding time is cooled to the temperature required for fermentation using
two chronological heat exchangers - the first using the coming medium as the cooling
source and the second using water
 In continuous process high temperature is used which reduce the holding time and nutrient
loss
 The necessary Del factor required may be attained by the proper temperature and holding
time which decrease the amount of nutrient loss
 Additionally, a continuous process engage heating of small amount of medium and cooling
of small amount of medium which is very less in contrast with batch system
 There are two types of continuous sterilizer:
1. The indirect heat exchanger

2. The direct heat exchanger (steam injector)

Continuous Sterilizer
Indirect Heat Exchanger Double spiral type
The most appropriate indirect heat exchanger is double-spiral type -consist of two sheets of high-
grade stainless steel, they are mould around central axis in such a way that they form a double
spiral, as shown in Fig. 5.8.
Steam and Medium passed through two different plates in opposite direction to attain
sterilization temperature.
This sterilizer is also use for cooling of the medium after proper holding time.
Incoming unsterile medium ispartially heated which itself is a cooling agent for medium which is
there in the sterilizer.
The major advantages of the spiral heat exchanger are
A. There is less chances of contamination between medium and liquid used for
cooling or steam, as they are separately moving in a compartment formed by stainless steel plates
with gasket seals at the end of the plates.
B. Exchanger will be clean by the steam or the liquid used for cooling and continues
movement of the media, so that there is less chances of sedimentation, fouling,and ‘burning on’.
Alternate plates of heat exchanger allow two different liquid or steam to circulate through them
in opposite direction.

Two plates divided by gasket; problem with this gasket can lead to cross contamination of
various streams.
This sterilizer is useful for completely soluble media as any suspended solids can block the
plates.
Ability of this Sterilizer increases by adding extra plates to it.
In continuous stem injector, steamdirectly inject into the unsterile broth.
Advantages Indirect heat exchanger
(i) Immediate) heating up times

(ii) Media containing solids sterilized by this exchanger
(iii) Lessinvestment
(iv) Easy to maintain and clean
(v) Efficient in using steam
Disadvantages Indirect heat exchanger is
(i) Heating may cause foams

(ii) Steam is in direct contact with medium, so medium should be enough
concentrated and steam should be free from any agent responsible for
anticorrosion
B. Direct Heat Exchanger
In direct heat exchanger medium heated with the help of steam and cooled by sudden expansion
of the medium in vacuum compartment.
Cooling happensalmostinstantaneously.
Fig. 5.10 Continuous sterilization system(direct steam injection)

Fig. 5.11 The continuous sterilization system (spiral heat exchangers) is shown below
Hot water passes through system for the sterilization of the plant before sterilization of the
medium.
Steam used to sterilize pipe work and fermentor.

Heat is conserved by using incoming media, which will cool sterile medium, which in turn get
preheated before reaching the sterilizer.
Advantages of continuous steam injector
1 very short heating up time

2 it may be used for media containing suspended solids
3 low capital cost
4 easy cleaning and maintenance
5 high seam utilization efficiency
Disadvantages of continuous seam injector
1 foaming may occur during heating

2 direct contact of medium with steam require that allowance be made for condensate
dilution and require ‘clean’ steam, free from anticorrosion additives

References
 Principles of Fermentation Technology: (2nd edition, by Peter F. Stanbury, Allan Whitaker and Stephen J.
Hall, Butterworth-Heinemann, An imprint of Elsevier Science.)
 Industrial Microbiology: (By Casida L. E.New Age international (P) ltd publications)
 A Text Book of Industrial Microbiology: (2nd edition By WulfCrueger&AnnelieseCrueger)
 Biotechnology: Food Fermentation Microbiology, Biochemistry & Technology Vol. 1 & 2:(By V.K. Joshi &
Ashok Pandey)
 Manual of Industrial Microbiology and Biotechnology: (2nd Edition by Arnold L. Demain and Julian E.
Davies, Ronald M. Atlas, Gerald Cohen, Charles L. Hershberger, Wei-Shou Hu, David H. Sherman, Richard
C. Willson and J. H. David Wu)
 Industrial Microbiology-An introduction: By Michael J. Waites, Neil L. Morgan, John S. Rockey and Gary
Higton)
 Comprehensive Biotechnology-The Principles, Applications and Rugulations of Biotechnology in
Industry, Agriculture and Medicine: (By Mrray Moo Young)
 Fermentation Technology : Up Stream Fermentation Technology- Vol-I: (By H. A. Modi-Pointer
Publications)
 Fermentation Technology : Down Stream Fermentation Technology- Vol-II: (By H. A. Modi-Pointer
Publications)
 Industrial Microbiology by Prescott and Dunn's: (4th edition, edited by Gerald Reed, CBR publications)
 Fermentation Technology: (By M.L. Srivastava, NAROSA publications)
 Industrial Microbiology: (By A.H. Patel)
 International student edition: Microbiology- A laboratory Manual: (4th edition. By James G.
Chappuccino& Natalie Sherman)
 Bacteriological Techniques: (By F.J. Baker)
 Introduction to Microbial Techniques: (By Gunasekaran)
 Mannual of Industrial Microbiology and Biotechnology: (2nd Edition by Arnold L. Demain and Julian E.
Davies, Ronald M. Atlas, Gerald Cohen, Charles L. Hershberger, Wei-Shou Hu, David H. Sherman, Richard
C. Willson and J. H. David Wu)
Web references
 http://www.homebrew.net/ferment/
 http://www.soyinfocenter.com/HSS/fermentation.php
 http://www.ensymm.com/pdf/ensymm_fermentation_abstract.pdf
 http://scialert.net/fulltext/?doi=jm.2007.201.208
 http://aem.asm.org/content/7/1/57.full.pdf
 http://www.slideshare.net/yongkangbirdnest/lecture-4-sterilization
 http://www.ars.usda.gov/research/publications/publications.htm?seq_no_115=140721
 http://www.scribd.com/doc/30706834/Fermentation-Design
 http://www.wiley-vch.de/books/sample/3527318194_c01.pdf
 http://www.engineersirelandcork.ie/downloads/Biopharmaceuticals%2020Jan09%20-%202%20-
%20Ian%20Marison%20DCU.pdf
 www.yobrew.co.uk/fermentation.php
 http://bioscipub.com/journals/bbb/pdf/19-24.pdf
 http://gertrude-old.case.edu/276/materials/web/immobilizedenzymereview.pdf

 http://download.bioon.com.cn/upload/month_0902/20090223_b809d1c59ba2a6e2abfdJtWiJOiFDm02.attach.
pdf
 http://bioprocess-maulik.blogspot.in/2007/07/design-of-industrial-fermentation.html
 http://hsc.csu.edu.au/biology/options/biotechnology/3051/biotechnologyPart3.html
 http://www.rsc.org/ebooks/archive/free/BK9780854046065/BK9780854046065-00001.pdf
 http://www.biotech.upm.edu.my/academics/On%20Line%20Note/Bioprocess/BTK%205301/Lect6%28Inocu
lum%20Preparation%20and%20Development%29.pdf
 http://www.biotechresources.com/services-strain.shtml
 http://www.idosi.org/wjc/4%281%2909/14.pdf
 http://cheserver.ent.ohiou.edu/Paper-gu/DualFeed.pdf

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NMEICT-MHRD (Govt.

of India) Project on - Creation of e-Contents on Fermentation Technology

Module – 15: Batch and continuous sterilization process

A number of factors influence the success of heat sterilization

1 The number and types of microorganisms present

N is the number of viable organisms present,

t is sterilization treatment time,

On integration of equation (5.1), the following expression obtained….

Project control No: RE-02091011297, Christ College, Rajkot, Gujarat, India

Nt/No = e-kt (2)

No is the number of viable organisms present initially

Nt is the number of viable organisms present after a period treatment, t

On taking natural logarithms, equation (5.2) reduced to

ln(Nt/ No) = -kt (3)

kt= ln (N0/ Nt) (4)

Project control No: RE-02091011297, Christ College, Rajkot, Gujarat, India

Project control No: RE-02091011297, Christ College, Rajkot, Gujarat, India

On taking natural logarithm

Plot of ln k versus 1/T gives straight line

Now lnNt/No = -kt

Therefore ln No/Nt = A. t. e –E/RT

But from above equation ln No/Nt = A. t. e –E/RT

It should be remembered that B. stearotheromphilusis not always adopted as the design

Project control No: RE-02091011297, Christ College, Rajkot, Gujarat, India

During sterilization,the nutrient value of the medium decreasesdue to following Reactions.

Interactions between nutrient components of the medium

 Maillard-type browning reaction discoloration of the medium as well as deterioration

Degradation of heat labile components

Project control No: RE-02091011297, Christ College, Rajkot, Gujarat, India

Advantages of continuous sterilization over batch sterilization

1. Betterprotectionof medium value

Advantages of batch sterilization over continuous sterilization

The Design of Batch Sterilization Processes

1. How much temperature of the fermentation medium is increased during heating or

Project control No: RE-02091011297, Christ College, Rajkot, Gujarat, India

▼overall = ▼heating +▼holding +▼cooling

Batch sterilization Methods

Advantages of a separate medium sterilization vessel

Project control No: RE-02091011297, Christ College, Rajkot, Gujarat, India

Disadvantages of a separate medium sterilization vessel

The design of continuous sterilization process

1. The indirect heat exchanger

Project control No: RE-02091011297, Christ College, Rajkot, Gujarat, India

Indirect Heat Exchanger Double spiral type

The major advantages of the spiral heat exchanger are

Project control No: RE-02091011297, Christ College, Rajkot, Gujarat, India

Ability of this Sterilizer increases by adding extra plates to it.

In continuous stem injector, steamdirectly inject into the unsterile broth.

Advantages Indirect heat exchanger

(i) Immediate) heating up times

Disadvantages Indirect heat exchanger is

(i) Heating may cause foams

B. Direct Heat Exchanger

Fig. 5.10 Continuous sterilization system(direct steam injection)

Project control No: RE-02091011297, Christ College, Rajkot, Gujarat, India

Steam used to sterilize pipe work and fermentor.

Project control No: RE-02091011297, Christ College, Rajkot, Gujarat, India

Advantages of continuous steam injector

1 very short heating up time

Disadvantages of continuous seam injector

1 foaming may occur during heating

Project control No: RE-02091011297, Christ College, Rajkot, Gujarat, India

Project control No: RE-02091011297, Christ College, Rajkot, Gujarat, India